Background Retinal degenerative diseases manifest with heterogeneous phenotypes, suggesting that one single therapeutic approach may not be applicable to all. Different modalities of intervention (in some cases in combination) could be targeted to specific retinal diseases based on known mechanisms of pathogenesis. We are testing gene therapy, small molecules, and retinal reconstruction to identify novel modalities to treat retinal degenerative diseases. All strategies are guided by the knowledge acquired through other projects of the molecular mechanisms of photoreceptor development, homeostasis, and disease (see EY000450-73-75). - Gene therapy Pre-clinical studies in collaboration with Z. Wu and T. Li (NNRL, NEI) are well underway for the treatment of retinitis pigmentosa (RP) caused by mutations in the RPGR and RP2 genes and of Leber congenital amaurosis (LCA) caused by CEP290 mutations. We focused on animal models and genetic characterizations, and Z. Wu's group worked on AAV-vector generation and gene therapy. Mouse and human RPGR-AAV vectors were tested in the RPGR-/- and rd9 mutant mouse models. A third mouse model with faster degeneration was also generated by crossing to a strain with a different background. Human and mouse vectors showed an effect in the RPGR-/- model at 18-24 months post-administration. Upon injection of the AAV8 human RP2 vector in RP2-null mice, cone function was rescued at 4 months post-administration and persisted up to 18 months. AAV8-RP2 effect on rod function could not be evaluated because of the rod degeneration kinetics with stabilization at one month of age. The large size of CEP290 cDNA exceeds the packaging limit of AAV vector. Among several AAV vectors that we tested, we recently identified one vector that is able to preserve retinal structure and function of the Cep290 mutant mouse model. A patent application has been filed (1). - Small molecule screening We are using zebrafish, embryonic and induced pluripotent stem cells (ESCs and iPSCs) and in vitro cell-based cultures to develop protocols for high throughput screens (HTS) of small molecules. One of our aims is to identify small molecules that facilitate the generation of rod and cone photoreceptors in zebrafish or from hESCs and hiPSCs expressing rod- and cone-specific transgenic reporter lines. We selected one cone-specific candidate molecule resulting from screening of the NCI Diversity II set (2000 compounds) and The Spectrum Collections (3000 compounds) chemical libraries using two zebrafish transgenic reporter lines (Rho-GFP and TalphaCP-GFP). We are further investigating the effects of the candidate compound and the developmental and/or homeostatic pathways affected. Rod or cone photoreceptors produced from hESCs or hiPSCs are a potential renewable source for cell-based therapy. However, lengthy differentiation protocols and low yield make their use currently unpractical. We are testing small molecule libraries using hES and hiPS cell lines with fluorescent reporter constructs driven by photoreceptor-specific promoters to identify compounds that facilitate the differentiation of high quality and pure transplantable photoreceptor cells with moderate cost and effort. - Use of iPSCs and ESCs to develop therapies Although animal models are an essential resource to study retinal development, homeostasis, aging/disease and to test novel therapies, they not always recapitulate the features of the human retina. Three-dimentional organoids generated from floating aggregates of hESCs are a novel tool for elucidating the mechanism underlying retinogenesis and disease. We generated a hESC line expressing a reporter GFP in rod and cone photoreceptor precursors under control of the cone-rod homeobox (CRX) gene promoter. GFP pattern of expression recapitulated that of endogenous CRX in the differentiating retinal organoids that also expressed markers of rod and cone photoreceptors and of other retinal neurons and Mller glia. Time-course gene expression profiling by RNA-seq of GFP+ flow sorted photoreceptors was performed to monitor the developmental steps in vitro. The gene expression signature revealed that CRX-GFP+ cells derived from hESCs are similar to human fetal photoreceptors, validating the organoids as in vitro models for studies of the human retina. Several patient and control iPSCs have been generated and are being characterized to be used to study the effect of gene mutation and of targeted therapies on photoreceptors. The lengthy differentiation protocols to generate photoreceptors from human pluripotent stem cells pose a limitation also to their use as pre-clinical testing systems. To overcome this limitation, we focused on mouse induced pluripotent stem cells for proof-of-concept preclinical studies that can be further evaluated on human cells at a later stage of therapy development. We generated iPSCs from the Cep290/rd16 mutant mouse and differentiated them into retinal organoids. We found that the architecture and gene expression of the Cep290/rd16 iPSC-derived retina were abnormal compared to WT controls. The retinal differentiation assay will be employed to test the efficacy of AAV vectors encoding different domains of CEP290 for gene therapy.